1. Field of the Invention
The present invention relates to a three-dimensional confocal microscope system and, more specifically, to improvements made in order to consistently obtain sliced confocal images of samples.
2. Description of the Prior Art
With a confocal microscope, it is possible to obtain sliced images of a sample without thinly segmenting the sample, and to construct a precise three-dimensional image from these sliced images. As a result, the confocal microscope is used for physiological reaction observation or for morphological observation of live cells in the fields of biology and biotechnology, or for the surface observation of LSI devices in the semiconductor market (see patent document 1, for example).
Note that in these sample observation modes, ultra-deep images (also referred to as omnifocal images) in full focus across the sample are required in some cases. In those instances, multiple sliced images at the respective focal positions of the sample are first obtained, then the images are subjected to image processing for synthesis so that an ultra-deep image is ultimately obtained (see non-patent document 1, for example).                Patent Document 1        Japanese Laid-open Patent Application 2002-72102        Non-Patent Document 1        A catalog introducing the VK-9500 ultra-deep 3D color imaging/shape measurement microscope from Keyence Corporation (checked out at the following URL through an Internet search on Aug. 14, 2003)        <URL:http://www.keyence.co.jp/microscope/product/VK9500/index.html>        
FIG. 1 is a block diagram illustrating the configuration of the confocal microscope described in patent document 1.
Video rate camera 1, confocal scanner 2, microscope 3, actuator 4 and objective lens 5 are all aligned on the same optical axis. Confocal scanner 2 comprises a Nipkow disk having a multitude of pinholes and a microlens array associated with this disk. The confocal scanner is thus of a compact add-on type which is composed of a simple optical system, employing the Nipkow disk method.
This confocal scanner 2 is mounted on the camera port of microscope 3. Using laser light, the confocal microscope inputs images of the sample through objective lens 5, actuator 4 and microscope 3 to confocal scanner 2. Confocal scanner 2 receives confocal images of the sample and inputs them to video rate camera 1.
FIG. 2 is a timing chart of various signals dealt with by the confocal microscope illustrated in FIG. 1. Video rate camera 1 converts the confocal images to video signal 101 and inputs the signal to the signal input terminals of confocal scanner 2 and synchronization interface box 9 and to the video input terminal of image processing unit 6. Confocal scanner 2 is responsible for the rotational synchronization control of the Nipkow disk in synchronization with video signal 101.
In an application where a videotape deck is employed in image processing unit 6, the videotape deck simultaneously records both video signal 101 being input from the video input terminal and start signal 103 being input from the audio input terminal on long-playing videotape. On the videotape, confocal images in a real-time variation state and the timings to start scanning the focal positions of objective lens 5 are recorded simultaneously.
Synchronization interface box 9 selects either the even-numbered pulse train or odd-numbered pulse train of video signal 101 to produce internal signal A. Arbitrary waveform generator 7 generates trigger signal 102 which is a high-state pulse signal, there inputs the trigger signal to the trigger input terminal of synchronization interface box 9 so that the trigger signal is used for the timing to start scanning the focal plane in question.
Synchronization interface box 9 produces internal signal B in synchronization with the falling edges of trigger signal 102. This internal signal B has a high-state pulse width of approximately 35 ms, which is slightly wider than the one defined by the video rate of video rate camera 1. Synchronization interface box 9 generates start signal 103 by performing logical AND operation on the inverted signal of internal signal A and internal signal B, and inputs the start signal to the synchronization input terminals of image processing unit 6 and arbitrary waveform generator 7.
Image processing unit 6 starts image capture in which video signal 101 is converted to image data and recorded in synchronization with the rising edge of start signal 103 which is input through the synchronization input terminal. According to video signal 101 input through the signal input terminal, synchronization interface box 9 synchronizes all of the rotational synchronization control of the Nipkow disk by confocal scanner 2, the timing for image processing unit 6 to start obtaining video signals, and the timing for the optical control system to start scanning the focal positions of the objective lens. Arbitrary waveform generator 7 starts scanning the focal positions of objective lens 5 in synchronization with the rising edge of start signal 103, using the optical control system. In addition, arbitrary waveform generator 7 generates scanning signal 104 and inputs it to controller 8. Scanning signal 104 is a sawtooth signal that linearly rises from a low state to a high state over a specified period of time. Controller 8 inputs scanning signal 104 to actuator 4. Actuator position signal 105 is the positional signal of an actual actuator which after fully ramping up, falls back to the original level at one stroke, followed by an overshoot, where the period of the overshoot corresponds to a dead band.
Actuator 4 is installed between the objective lens revolver of microscope 3 and objective lens 5. The length of actuator 4 in the focal-point direction of images is changed by piezoelectric drive in proportion to the level of scanning signal 104, in order to control the focal position of objective lens 5. The confocal microscope obtains sliced images of the sample by scanning the focal plane thereof according to scanning signal 104.
According to the system configuration described above, the rotational synchronization control of the Nipkow disk, the timing for the image processing unit to start obtaining video signals, and the timing for the optical control system to start scanning the focal positions of the lens are all synchronized with the video signal. Consequently, the positional accuracy of confocal images increases, thereby eliminating variations in the time taken to obtain each sliced image when multiple sliced images are obtained and providing highly reliable sliced images.
In the conventional confocal microscope, however, a ramp wave (right-angled triangular wave) is used as the scanning waveform for the actuator to scan the focal plane of the objective lens. For this reason, enormous acceleration takes place in the actuator at each turn-around point of a waveform and a prolonged period of time is required for such acceleration to stabilize. Since accurate images cannot be obtained during this stabilization time, the conventional confocal microscope has been hampered in that the number of effective images that can be used for observation are decreased.
Furthermore, such enormous acceleration can result in vibration and resonance may take place if the vibration frequency matches the natural vibration frequency of the actuator itself and the microscope enclosure. This would lead to the problem that the surfaces of the sample become unstable and therefore cannot be correctly observed.